Adenosine deaminase resistant antiviral purine nucleosides and method of preparation

ABSTRACT

The preparation of (±)-9-[α-(2α,3β-dihydroxy-4α-(hydroxymethyl)cyclopentyl)]-6-substituted purines: ##STR1## and (±)-3-[α-(2α,3β-dihydroxy-4α-(hydroxymethyl)cyclopentyl)]-7-substituted-v-triazolo[4,5d]pyrimidines: ##STR2## and their derivatives wherein R is amino, mercapto, methylmercapto, hydroxy, halogen, or substituted amino: ##STR3## wherein R&#39; and R&#34; may be the same or different and are of hydrogen, methyl, ethyl, propyl or phenyl. The preparation of the single intermediate from which either of these series of compounds may be synthesized is also disclosed. The compounds exhibit antiviral and antitumor activity. Acid salts and esters of the purine nucleosides have also been prepared.

The invention described herein was made in part in the course of workunder a grant or award from the Department of Health, Education andWelfare.

This application is a continuation-in-part of my copending applicationSer. No. 766,947, filed Feb. 9, 1977, now U.S. Pat. No. 4,138,562,issued Feb. 6, 1979.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to the preparation of certain chemotherapeuticmaterials useful in the treatment of viral infections and tumorsassociated with viruses. More specifically, the invention is directed tothe preparation of adenosine deaminase resistant antiviral purinenucleosides.

2. Description of the Prior Art

The antiviral nucleoside 9-β-D-arabinofuranosyladenine (ara-A) was firstsynthesized in a program designed to produce anticancer agents. Recentinterest in the promising antiviral activity of ara-A has beenextensively reviewed. Broad spectrum activity of ara-A against DNAviruses and significant therapeutic activity of ara-A againstexperimental herpes simplex keratitis and herpes simplex and vaccinialencephalitis has been reported. A major liability in the use of ara-Alies in the fact that the nucleoside is rapidly deaminated by a commonlyoccurring enzyme, adenosine deaminase. Deamination of ara-A renders itmuch less effective and high doses of the drug are required at frequentintervals. Although the deamination product,9-β-D-arabinofuranosyl-hypoxanthine (ara-H), is also active against DNAviruses, it is considerably less active than ara-A. A major effort tocircumvent the deamination problem employs the use of ara-A incombination with adenosine deaminase inhibitors such as deoxycoformycinor erythro-9-(2-hydroxy-3-nonyl)adenine. This approach presents aproblem in that the Food and Drug Administration is reluctant to approveand physicians are reluctant to prescribe a compound that inhibits anenzyme with a normal body function. A more desirable approach to thedevelopment of a more active antiviral or antitumor agent, followed byapplicant, involves the use of a deamination resistant ara-A derivative.The carbocyclic ara-A analogs described herein circumvent the majordisadvantage of ara-A because they are completely resistant todegradation by adenosine deaminase.

SUMMARY OF THE INVENTION

The invention is directed to the synthesis of the intermediate: ##STR4##wherein X is a halogen, and the synthesis from it of either of thepurine nucleosides: ##STR5## wherein R is amino, hydroxyl, mercapto,methylmercapto or substituted amino: ##STR6## wherein R' and R" areeither the same or different and are hydrogen, methyl, ethyl, propyl orphenyl.

BRIEF DESCRIPTION OF THE DRAWING

The single FIGURE of the accompanying drawing is a flow diagram showingthe synthesis of the purine nucleosides.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The recent description of an unequivocal route to2-azabicyclo[2.2.1]hept-5-en-3-one (1, referring to the correspondingnumber on the flow sheet) (Jagt et al., J. Org. Chem., 39, 564 (1974))offers a unique starting point for the synthesis of carbocyclicaminonucleosides of known geometric configuration. Acidic hydrolysis ofthis compound to cis-4-aminocyclopent-2-ene carboxylic acidhydrochloride, followed by esterification of the carboxyl function inrefluxing methanol and subsequent acetylation of the amino group inacetic anhydride-pyridine, gives methyl(±)-cis-4-acetamidocyclopent-2-ene carboxylate (2). Reduction of themethyl ester of 2 gives, after acetylation, acetate 3. Epoxidation ofacetate 3 is stereoselective due to the syn-directing allylic amidegroup, giving only the cis-epoxide 4. Hydrolysis of the easilysynthesized epoxide 4 and subsequent acetylation gives a mixture of 5, 6and 6a.

When the major isomer,(±)-4α-acetamido-2β,3α-diacetoxy-1α-cyclopentanemethyl acetate (5) issubjected to mild acidic hydrolysis, amine 7 is formed, since acylmigration to the adjacent cis-hydroxyl facilitates hydrolysis of theacetamide. Amine 7, a hygroscopic gum, is immediately condensed with5-amino-4,6-dihalopyrimidine, giving intermediate5-amino-4N-[2α,3β-dihydroxy-4α-(hydroxymethyl)cyclopent-1α-yl]amino-6-halopyrimidine(8). This intermediate, when treated with the ring closing reagentsleads to the preparation of two series of purine nucleoside analogs, 9and 11.

The nucleoside analogs (called carbocyclic arabinosyl adenosine analogs)9 and 11 can be converted to a series of antiviral and antitumor agentsby reaction with substituting agents for adding a substituent in the6-position of 10 or the 7-position of 12. These substituting agents (R:)are selected from the group consisting of amino, mercapto,methylmercapto, hydroxy and substituted amine. The antiviral compoundsare represented by structures 10 and 12.

The invention is illustrated by the following examples:

EXAMPLE 1

The intermediate,5-amino-4N-[2α,3β-dihydroxy-4α-(hydroxymethyl)cyclopent-1α-yl]amino-6-chloropyrimidine(8), was prepared as follows:

Methyl (±)-cis-4-acetamidocyclopent-2-enecarboxylate (2).2-Azabicyclo[2.2.1]heptan-3-one (Jagt et al., J. Org. Chem., 39, 564(1974)) (64.2 g, 0.588 mole) was dissolved in 5% HCl (2500 ml) and thesolution stirred at room temperature (RT) for 3.5 days. A small amountof gummy solid was filtered off, and the filtrate was cooled (ice bath)while sufficient 6 N NaOH (ca. 500 ml) was added to give pH 1.0. Thepale yellow solution was evaporated to dryness (<50%, 0.5 mm). Theresidue was azeotroped with PhH--MeOH, dried at 0.1 mm/RT, and thenrefluxed in dry MeOH (1 liter) for 18 hrs. The NaCl was filtered off andwashed with additional MeOH. The MeOH filtrate-wash was evaporated todryness and the residual yellow syrup dissolved in pyridine (500 ml).Acetic anhydride (300 ml) was added to the cooled (ice bath) solution.The solution was allowed to come to RT. After 1.0 hr. at RT, thesolution was evaporated (<50°, 0.5 mm) to dryness. The residue wasdissolved in CH₂ Cl₂ (500 ml) and extracted with sat'd NaHCO₃ (3×200ml), sat'd NaCl (50 ml), and dried (CaSO₄). Evaporation and azeotropingwith toluene (3×200 ml, to remove pyridine) left a yellow syrup (103.5g) which solidified within a few minutes with the generation ofconsiderable heat. The nmr spectrum of this off-white solid wasidentical with that of an analytical sample. Sublimation (70°-80°, 0.003mm) gave methyl-cis-4-acetamidocyclopent-2-enecarboxylate as whitecrystals (96.1 g, 89%); mp 66°-67°(±)-cis-4-Acetamidocyclopent-2-enemethyl Acetate (3). A mixtute of CaCl₂(31.8 g, 0.286 mole) and NaBH₄ (21.7 g, 0.572 mole) in drytetrahydrafuran (THF) (freshly distilled from lithium aluminum hydride)(LAH), 600 ml) was stirred at RT for 1.0 hr. A solution of 2 (35.0 g,0.191 mole) in dry THF (500 ml) was added all at once. The resultingmixture was stirred at RT for 18 hrs. It was then cooled (ice bath) andice-H₂ O (700 ml) added dropwise (much effervescence at first). Cold 6NHCl (110 ml) was then added (to a pH of 1.5) and the resulting clearsolution stirred at RT for 1.0 hr. Evaporation, azeotroping with MeOH(4×500 ml), then with pyridine (2×500 ml) gave a mixture of white solidand pale yellow syrup. Pyridine (250 ml) was added, and the insolubleinorganics filtered off. Acetic anhydride (250 ml) was added to thepyridine filtrate and stirring continued at RT for 18 hrs. Afterevaporation, MeOH (250 ml) was added to the residual syrup and theresulting solution refluxed for 10 min. After evaporation of the MeOH,the residue was stirred with CH₂ Cl₂ (500 ml)-H₂ O (250 ml) whilesufficient solid NaHCO₃ was added to make the aqueous layer basic. Thelayers were separated and the aqueous layer was extracted withadditional CH₂ Cl₂ (2×250 ml). The combined CH₂ Cl₂ layers were dried(CaSO₄) and evaporated. The residue was azeotroped with toluene (3×250ml), leaving a yellow oil (39.1 g); pmr almost identical to that of ananalytical sample. Distillation gave a colorless syrup (36.7 g, 98%), bp132°-134° (0.04 mm), which solidified on standing to white crystals, mp62°-63°. Sublimation of such a sample (60°, 0.1 mm) gave an analyticalsample of 3 as white crystals: mp 62°-63°.

(±)-4α-Acetamido-2α, 3α-epoxycyclopentane-1α-methyl Acetate (4). Asolution of 3 (36.7 g, 0.186 mole) and m-chloroperbenzoic acid (37.8 g,85%, 0.186 mole) in CCl₄ (700 ml) was refluxed for 2.0 hrs. The solutionwas concentrated to 200 ml and CH₂ Cl₂ (500 ml) added. This solution wasextracted with sat'd NaHCO₃ (150 ml), dried (CaSO₄), and evaporated,leaving 4 as a yellow oil (40.8 g) which solidified on standing.

(±)-4α-Acetamido-2β, 3α-diacetoxy-1α-cyclopentanemethyl Acetate (5) and(±)-4α-Acetamido-2α, 3β-diacetoxy-1α-cyclopentanemethyl Acetate (6). Asolution of crude 4 (7.42 g, 34.8 mmoles) in 2% H₂ SO₄ (450 ml) waswarmed (steam bath) for 1.0 hr. A small amount of gummy solid wasfiltered off (mostly m-chlorobenzoic acid contaminating 4). The pH ofthe cooled filtrate was adjusted to 7 (indicator paper) with 6 N NaOH.The H₂ O was evaporated and the residue dissolved in pyridine (2×200 ml)and evaporated. The residual syrup was dissolved in Ac₂ O (100ml)-pyridine (200 ml) and stirred at RT overnight. After evaporation,the residue was dissolved in CH₂ Cl₂ (250 ml), extracted with sat'd(NaHCO₃ (25 ml), and dried (CaSO₄). Evaporation, followed azeotropingoff pyridine with toluene, left brown syrup (9.61 g). Crystallizationfrom EtOAc gave 5 as white prisms (5.77 g, 53%), mp 137.5°-138.5°.

The mother liquors from crystallization of 5 contained an approximately1:1 mixture of 5 and 6 (from NH resonances in pmr spectrum). Althoughsome slight separation appeared on the (5% MeOH-CHCl₃, silica gel),column chromatography of the mother liquor contents on silica gel (250g) with elution by 1% MeOH--CHCl₃ gave only a slight enrichment of theearly fractions in the minor isomer (about 60:40 by pmr). The mixture of5 and 6 (3.30 g, 10.5 moles) was dissolved in 2 N HCl (100 ml) andmaintained at 70° (oil bath) for 1.0 hr. The solution was evaporated todryness. The residue was dissolved in H₂ O and the solution stirredbriefly with IRA-400(OH⁻) resin (30 ml). The solution (presumed to be7+6a) was passed slowly through a column of IRA-120(⁺) resin (60 ml).Elution of the column with H₂ O and azeotroping with abs. EtOH produced6a as a colorless syrup (1.13 g, 5.97 mmoles, 17% from 4). The syrup wasreacetylated in Ac₂ O-pyridine (as above), giving 6 as a colorless syrup(1.58 g, 14% from 4).

(±)-4α-Amino-2β, 3α-dihydroxy-1α-cyclopentanemethanol (7). A solution of5 (3.37 g, 10.7 mmoles) in 2 HCl (100 ml) was maintained at 70° (oilbath) for 1.0 hr. The solution was evaporated to dryness and the residuedissolved in MeOH (100 ml) and stirred briefly with IRA-400(OH⁻) resin(25 ml). Evaporation left 7 as a viscous syrup which could not besolidified and turned yellow on standing. Since 7 appeared to carbonateon exposure to air, it was used immediately.

5-amino-4N-[2α,3β-dihydroxy-4α-(hydroxymethyl)cyclopent-1α-yl]amino-6-chloropyridimide(8). A solution of 7 (syrup from hydrolysis of 10.7 mmoles of 5),5-amino-4,6-dichloropyrimidine (3.51 g, 21.4 mmoles), and triethylamine(7.5 ml, 53.5 mmoles) in 1-BuOH (50 ml) was refluxed under N₂ for 24hrs. The solution was evaporated to dryness and the residue partitionedbetween H₂ O (80 ml) and CHCl₃ (40 ml). The aqueous layer was separatedand extracted with additional CHCl₃ (3×10 ml). The combined CHCl₃ layersshowed only 5-amino-4,6-dichloropyrimidine on tlc. The aqueous layer wasstirred briefly with IRA-400(OH⁻) resin (18 ml). The H₂ O was thenevaporated and the residue dried by azeotroping with abs. EtOH, givingchromatographically homogeneous 8 as a pale yellow glass (3.4 g,contains EtOH). Such a sample was sufficiently pure for use in thefollowing reactions. Two recrystallizations of such a sample from abs.EtOH gave 8 as an off-white powder, 72% from 5: mp 184°-186°.

EXAMPLE 2

The purine nucleoside analog (±)-6-Chloro-9-[2α,3β-dihydroxy-4α-(hydroxymethyl)cyclopent-1α-yl]purine (9) was preparedas follows:

A solution of crude 8 (ca. 4.8 mmoles) in diethoxymethyl acetate (20 ml)was stirred at RT overnight and then at 100° (oil bath) for 1.0 hr. Thesolution was evaporated and then azeotroped with abs. EtOH and dried at0.05 mm for 2 days. The residual brown syrup (3.4 g) still smelled likediethoxymethyl acetate; tlc shows several spots at R_(f) greater thanthat of 8 or 9. The syrup was stirred vigorously with 0.5 N HCl (120 ml)at RT for 30 min. The resulting solution was adjusted to pH 7.8 (meter)by addition of IRA-400 (OH⁻) resin evaporation left while solid (0.98 g,72%), which tlc showed to be chromatographically homogeneous 9. Ananalytical sample of 9 was prepared by two recrystallization of such asample from abs. EtOH, giving white clusters of needles: mp 210°-212°dec.

EXAMPLE 3

The amino substituted derivative (10a) of the -6-substituted purine 10was prepared from the intermediate 8 as follows:

(±)-9-[α-(2α, 3β-Dihydroxy-4α(hydroxymethyl)cyclopentyl]adenine(C-ara-A) (10a). A solution of crude 8 (ca. 4.1 mmoles) indiethoxymethyl acetate (25 ml) was stirred at RT overnight and then at100° (oil bath) for 1.0 hr. Alternatively, triethyl orthoformate hasbeen used to close the ring. The solution was evaporated to dryness andthe residue shaken with NH₃ (1., 50 ml) in a stainless steel bomb at RTovernight. The NH₃ was allowed to evaporate and the residue dissolved in1 N HCl (100 ml) and stirred at 60° (oil bath) for 45 min. The solutionwas evaporated to dryness, the residue dissolved in MeOH and passedthrough a column of IRA-400(OH⁻) resin (20 ml). The MeOH eluent (250 ml)was evaporated, the tan solid residue (870 mg) was triturated with abs(EtOH, giving 10a as white powder (824 mg, 76%), mp 253°-255° dec.

EXAMPLE 4

The hydroxyl substituted derivative (10b) of the -6-substituted purine10 was prepared as follows:

(±)-9-[2α, 3β-Dihydroxy-4α-(hydroxymethyl)cyclopent-1α-yl]hypoxanthine(10b). A sample of 8 (2.0 mmoles) which had been treated withdiethoxymethyl acetate as described in Example 2 in the preparation of 9was then refluxed in 1N HCl (25 ml) for 3.5 hrs. The solution wasevaporated to dryness and the residue dissolved in H₂ O (25 ml). The pHwas adjusted to 5-6 by addition of IRA-400 (OH⁻) resin in smallportions. Evaporation, followed by azeotroping with abs. EtOH, leftchromatographically homogeneous 10b as a white powder (257 mg), mp220°-222° dec. Crystallization from MeOH gave white granules (247 mg,46%): mp 221.5°-223.5° dec.

EXAMPLE 5

The mercapto substituted derivative (10c) of the -6-substituted purine10 was prepared as follows:

(±)-9-[2α,3β-Dihydroxy-4α-(hydroxymethyl)cyclopent-1α-yl]-9H-purine-6-(lH)-thione(10c). A solution of 9 (310 mg, 1.09 mmoles) and thiourea (142 mg, 1.86mmoles) in 1-propanol (8 ml) was refluxed for 45 min., at which timewhite solid had precipitated. The mixture was cooled and the solidfiltered off and washed with 1-propanol (2×2 ml), giving 12 as whitepowder (237 mg, 77%), same melting characteristics and tlc as ananalytical sample. Crystallization from H₂ O gave an analytical sampleof 12 as white granules (185 mg): mp dependent of rate of heating,starts to dec. at ca. 270°, black fluid by ca. 280°.

EXAMPLE 6

The methylmercapto substituted derivative (10d) of the -6-substitutedpurine 10 was prepared as follows:

(±)-9-[2α,3β-Dihydroxy-4α(hydroxymethyl)cyclopent-1α-yl]-6-(methylthio)purine(10d). A mixture of crude 10c (174 mg, 0.616 mmole), methyl iodide (0.25ml), 1.0 N NaOH (0.62 ml), and H₂ O (2.0 ml) was stirred at RT for 4.0hrs. The resulting solution was evaporated to dryness and the residuechromatographed on a column of silica gel G (Brinkmann, 20 g, packed inCHCl₃). Elution with 5% MeOH--CHCl₃ and combination of the UV-absorbingfractions gave 10d as white powder (45 mg, 25%), chromatographicallyhomogeneous. Resolidification of such a sample from abs. EtOH gave ananalytical sample as white flakes: mp 232°:234°.

EXAMPLE 7

The amino substituted derivative (12a) of the -7-substituted pyrimidine12 was prepared as follows:

(±)-7-Amino-3-[α-(2α,3β-dihydroxy-4-(hydroxymethyl)cyclopentyl]-v-Triazolo[4,5d]pyrimidine(12a). To a cooled (ice bath) solution of 8 (526 mg, 1.91 mmoles) in 0.5N HCl (10 ml) was added NaNO₂ (159 mg, 2.30 mequiv). After 5 min., theice bath was removed and the solution was stirred at RT for 1.0 hr.Solid NaHCO₃ (420 mg) was added and the solution was evaporated todryness. The residue was shaken with NH₃ (20 ml) in a steel bomb at RTfor 20 hrs. After evaporation of the NH₃, the residue was solidifiedfrom H₂ O, giving white solid (294 mg, 58%), mp 258°-262° dec.

EXAMPLE 8

The cytotoxicity of C-ara-A was evaluated by growing P-388 mouselymphoid leukemia cells in the presence of either (C-ara-A) or ara-Ausing the method described by Almquist et al., J. Med. Chem., 16, 1396(1973). Both ara-A and C-ara-A exhibited LD₅₀ concentrations of 1×10⁻⁵M. In contrast to ara-A, the carbocyclcic analog C-ara-A is completelyresistant to deamination by adenosine deaminase. Thus, under conditionsin which ara-A is completely deaminated (1 μmole/min/unit of enzyme) bycalf intestinal adenosine deaminase (type III, Sigma) no detectabledeamination of C-ara-A was observed. In addition, C-ara-A did notinhibit the enzymatic deamination of either ara-A or adenosine.

EXAMPLE 9

C-ara-A was examined for in vitro antiviral activity against tworepresentative DNA-containing animal viruses by the quantitativedetermination of its ability to inhibit virus-induced cytopathogeniceffects (cpe) in infected cultures. The viruses employed in these assayswere herpes simplex virus (HSV) type 1 (strain HF) and vaccinia virus(VV) (Strain Lederle Chorioallantoic). Both viruses were propagated andassayed for infectivity in continuous-passage human epidermoid carcinomaof the larynx (HEp-2) cells. A virus rating (VR) was calculated for theactivity of C-ara-A against each virus by the use of a modification ofthe method of Ehrlich et al., Ann. N.Y. Acad. Sci., 130, 5 (1965)previously described by Sidwell et al., Proc. Soc. Exp. Biol. Med., 131,1226 (1969), except that triplicate cultures rather than duplicatecultures were employed for each assay. The results are shown in Table 1:

                  TABLE I                                                         ______________________________________                                        In Vitro Antiviral Activity of Carbocyclic Ara-A                                                              MED.sub.50.sup.b                              Challenge Virus  Virus Rating (VR).sup.a                                                                      (μg/ml)                                    ______________________________________                                        Herpes simplex virus, type 1                                                                   2.2            9.0                                                            3.5            2.8                                           Vaccinia virus   1.5            9.0                                                            1.7            9.0                                           ______________________________________                                         .sup.a Virus rating (VR): a weighted measurement of antiviral activity,       based on the in vitro inhibition of virusinduced cytopathogenic effects       (cpe) and the cytotoxicity exhibited by the drug, determined by a             modification of the method of Ehrlich et al. (supra). A VR≧1.0         indicates definite (+) antiviral activity; a VR of 0.5-0.9 indicates          marginal to moderate (±) antiviral activity; and a VR<0.5 indicates no     (-) apparent antiviral activity                                               .sup.b Minimum effective dose, 50% (MED.sub.50): the minimum drug dose        required for 50% inhibition of virusinduced cpe.                         

As can be seen, the carbocyclic analog of ara-A demonstrated highlysignificant antiviral activity against HSV and VV with VR's ranging from1.5 to 3.5. The approximate MED₅₀ for C-ara-A appears to be about 9g/ml.

Compounds 3 and 4 may also be prepared as benzoates or p-nitrobenzoate.

EXAMPLE 10

A chemotherapy experiment with carbocyclic ara-A against lethal HSV-1infections in mice was conducted by an independent research laboratory.The data are contained in the two following summary tables: Table 2shows the actual death patterns for treated and control mice and alsogives the average body weights of the animals on days 0, 7, 14 and 21.Table 3 summarizes the mortality and mean survival time data in a formatsuitable for publication.

It can be seen that 95 per cent of the virus-inoculated control micedied, with a mean survival time of 8.4 days for the dying animals. Thepositive control drug (ara-A) exhibited significant in vivo activity inthis model system by reducing the mortality down to 0 to 10 percent whenadministered at non-toxic dose levels of 125 to 250 mg/kg/day on the qd1-7 schedule. Likewise, the carbocylic analog of ara-A (C-ara-A) wasalso found to be highly effective in the treatment of these HSV-1infections in mice, reducing the mortality down to 0 to 10 percent whenadministered at the non-toxic dose levels of 112.5 to 450 mg/kg/day onthe same schedule. C-ara-A at 900 mg/kg/day was apparently not lethallytoxic for uninfected animals, but proved to be quite toxic for theHSV-infected mice. The mean survival time of dying animals in thislatter group was observed to be significantly reduced to 4.3 days. Theincrease in mean survival time of the one dying animal which was treatedwith C-ara-A at 450 mg/kg/day was not statistically significant. Thesedata indicate that carbocyclic ara-A is essentially as active as ara-Aagainst HSV-1 in vivo.

                                      TABLE 2                                     __________________________________________________________________________    The Effect of Carbocyclic Arabinosyladenine (C--Ara--A) Treatment on          Herpes                                                                        Simplex Virus, Type 1, Infections in Mice                                     __________________________________________________________________________    Host:                                                                             Random-bred Swiss mice, female, from Charles                                                              Drug Treatment:                                                                        C--Ara--A was suspended in 0.9%                                               NaCl                                     River Breeding Laboratories, Inc.    solution contaning 0.3%                                                       hydroxypropyl-                                                                cellulose (HPC) at four                                                       different con-                       Virus:                                                                            Herpes simplex virus, type 1, strain HS-123.                                                                       centrations so that a single                                                  daily dose                               Equal volumes of 10% mouse brain suspensions                                                                       volume of 0.01 ml/gm of body                                                  weight                                   from the 8th and 9th intracerebral (i.c.)                                                                          equaled 900, 450, 225, and 112.5                                              mg/kg/                                   mouse passages were pooled and diluted 1:3                                                                         day.                                     in phosphate-buffered saline (PBS). The mice                                                                       9-β-D-arabinofuranosyladenin                                             e (NSC                                   were infected by inoculating each mouse                                                                            404241; Ara--A) was suspended in                                              the                                      intraperitoneally (i.p.) with 0.3 ml of the                                                                        same manner so that a single                                                  daily dose                               1:3 dilution.                        volume of 0.01 ml/gm of body                                                  weight equaled                                                                250 and 125 mg/kg/day.               Period of Observation:  21 days.         The drugs were administered once                                              daily                                                                         for 7 days beginning 4 hours                                                  after virus                                                                   inoculation.                         Drug                                        Mortality:                        Dose            Death Pattern               No. Dead/                                                                            Avg. Body Wt.              mg/             No. of Days Post-Virus Inoculation:                                                                       Total No.                                                                            gm on Day:                 kg/day                                                                            Animal Group                                                                              3 4 5 6 7 8 9 10                                                                              11                                                                              12                                                                              13                                                                              14                                                                              15-21                                                                             of Mice (%)                                                                          0  7  14 21                __________________________________________________________________________    0   Virus controls, untreated                                                                       4 4 4 1 3 1 1   1     19/20                                                                             95 19.8                                                                             21.5                                                                             25 26.5              900 C--Ara--A + virus                                                                         2       1                   3/10                                                                              30 19.8                                                                             18.1   20.6                                                                   22.6                        C--Ara--A toxicity                      0/5 0  21.8                                                                             22.5   25.3                                                                   28                          controls                                                                  450 C--Ara--A + virus           1           1/10                                                                              10 19.7                                                                             19.3   23.3                                                                   23.8*                       C--Ara--A toxicity                      0/5 0  21.6                                                                             22.6   25.9                                                                   28.3                        controls                                                                  225 C--Ara--A + virus                       0/10                                                                              0  19.6                                                                             21.8   24.1                                                                   25.6                        C--Ara--A toxicity                      0/5 0  21.8                                                                             24.4   26.8                                                                   29.3                        controls                                                                  112.5                                                                             C--Ara--A + virus   1                   1/10                                                                              10 19.9                                                                             22.4   24                                                                     26.9                        C--Ara--A toxicity                      0/5 0  21.8                                                                             24.5   26.5                                                                   28.5                        controls                                                                  250 Ara--A + virus      1                   1/10                                                                              10 19.9                                                                             20.3   23.1                                                                   25.9                        Ara--A toxicity                         0/10                                                                              0  21.7                                                                             25.1   26.6                                                                   29.1                        controls                                                                  125 Ara--A + virus                          0/10                                                                              0  20 23.4   25.9                                                                   28.2                        Ara--A toxicity                         0/10                                                                              0  21.7                                                                             25.4   27.3                                                                   29.5                        controls                                                                  0   HPC, sham-injected i.p.                 0/10                                                                              0  23.5                                                                             26.8   29.7                                                                   31.7                        qd 1-7                                                                    0   PBS, sham-injected i.p.                 0/10                                                                              0  22 27.7   29.4                                                                   31.7                        1X                                                                        0   Normal, untreated                       0/15                                                                              0  22.5                                                                             27.1   29.0                                                                   31.2                        animals                                                                   __________________________________________________________________________     *Note: One animal, moribund on day 21, weighed 11.5 gm. This animal had       been sick 4-5 days.                                                      

                                      TABLE 3                                     __________________________________________________________________________    Effect of Treatment with Ara--A and with the Carbocyclic Analog of Ara--A     (C--Ara--A) on                                                                Mortality of Random-bred Swiss Mice Inoculated i.p. with Herpesvirus          hominis type 1                                                                       Drug   Virus-infected Animals Uninfected Animals                              Dose   Mortality   Mean Survival Time                                                                       Mortality                                Drug   (mg/kg/day).sup.1                                                                    (No. dead/total                                                                        %  (days).sup.2                                                                             (No. dead/total)                                                                       %                               __________________________________________________________________________    None   --     19/20    95 8.4         0/35    0                               Ara--A.sup.3                                                                         250    1/10     10*.sup.4                                                                        7.0         0/10    0                               "      125    0/10      0*                                                                              --          0/10    0                               C--Ara--A.sup.3                                                                      900    3/10     30**                                                                              4.3.sup.+ 0/5      0                               "      450    1/10     10*                                                                                11.0.sup.++                                                                            0/5      0                               "      225    0/10      0*                                                                              --         0/5      0                               "        112.5                                                                              1/10     10*                                                                              7.0        0/5      0                               __________________________________________________________________________     .sup.1 Drugs were administered i.p. once daily for seven days beginning 4     hours after virus inoculation.                                                .sup.2 Only animals dying on or before day 21 after virus inoculation wer     considered.                                                                   .sup.3 Ara--A = 9D-Arabinofuranosyladenine. C--Ara--A = Carbocyclic analo     of 9D-Arabinofuranosyladenine.                                                .sup.4 Probability that the observed increase in survivor number              (χ.sup.2test) or the observed increase or reduction in mean survival      time (ttest) was due to chance. *p <<0.0005; **p <0.001; .sup.+ p <0.005;     .sup.++ p >0.10. A pvalue of <0.05 was considered to indicate a               signficiant difference; a pvalue of <0.001 was considered to indicate a       conclusive difference.                                                   

Acid salts of C-ara-A are prepared as follows:

EXAMPLE 11

(±)-9-[2α, 3β-dihydroxy-4α-(hydroxymethyl)cyclopent-1α-yl]adeninehydrochloride. To 283 mg (1 mmole) of 10a was added 10 ml of 0.1Nhydrochloric acid. The solution was warmed to 602° for five minutes andthen evaporated in vacuo to a white solid. The solid was triturated withabsolute ethanol, (10 ml) at 0°-5°. The analytical product was obtainedby filtration of the triturate and gave a crystalline solid: 250 mg.

EXAMPLE 12

The crystalline sulphate salt was obtained when 10 ml of 0.1N sulfuricacid was added to 283 mg of 10a and the reaction mixture was treated asin Example 11 above.

EXAMPLE 13

The monophosphate ester of C-ara-A (10a): ##STR7## was prepared asfollows:

(±)-9-[2α, 3β-dihydroxy-4α-(hydroxymethyl)cyclopent-1α-yl]adenine4-dihydrogen phosphate (13a). A solution of trimethyl phosphate (6 ml),10a (567 mg) and phosphoryl chloride (0.37 ml) was stirred at -10° for 3hours and the clear solution was then poured into water (175 ml) and thesolution was stirred at rt for 1 hour, neutralized with ammoniumhydroxide and passed through a column of Amberlite (H⁺) resin (20 g).The column was washed with water, then lN ammonium hydroxide (500 ml).The basic eluent was evaporated to dryness and the residue was dissolvedin water. To this solution was added 6N hydrochloric acid (0.35 ml) andethanol (5 ml). The solution was cooled to -20° and allowed torefrigerate overnight. The pure product was removed by filtration andgave a white solid: yield, 368 mg; mp 230°-232°.

EXAMPLE 14

The monoesters of C-ara-A (10a): ##STR8## wherein R is H, CH₃, C₂ H₅, C₃H₇, C₄ H₉, C₅ H₁₁, C₆ H₁₃, C₇ H₁₅, C₈ H₁₇, C₉ H₁₉, C₁₀ H₂₁, C₁₁ H₂₃, C₁₂H₂₅, C₁₃ H₂₇, C₁₄ H₂₉, C₁₅ H₃₁ or C₁₆ H₃₃ were prepared by condensationof the corresponding acid chloride with 10a according to the generalprocedure exemplified by the preparation of the valerate ester (R=C₄H₉):

(±)-9-[2α, 3β-dihydroxy-4α-(hydroxymethyl)cyclopent-1α-yl]adenine4-valerate. The hydrochloride salt of 10a (73 mg) was suspended in 3 mlof dimethylformamide. Valeryl chloride (35 mg) in 1 ml ofdimethylformamide was added dropwise to the suspension, and the reactionmixture was stirred at room temperature overnight. After removal of thesolvent, the residue was dissolved in water and the aqueous solution waswashed with chloroform to remove excess valeryl chloride. The aqueoussolution was neutralized with sodium bicarbonate and extracted withethyl acetate. The organic layer was dried with anhydrous sodium sulfateand evaporated to yield a solid product (30 mg). The solid was purifiedon a prep-tlc (silica gel) plate developed with 20 percent methanol inmethylene chloride. The pure valerate was extracted from the silica gelwith the developing solvent and yielded a white powder; mp 151°-153°,mass spectra m/e 349 (M⁺.).

EXAMPLE 15

The formate ester (R=H) was prepared by dissolving 10a in anhydrousformic acid and allowing the resulting solution to stand for 4 days at5°. Isolation of the ester was as in Example 14 above.

It is apparent that many modifications and variations of this inventionas hereinbefore set forth may be made without departing from the spiritand scope thereof. The specific embodiments described are given by wayof example only and the invention is limited only by the terms of theappended claims.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of preparationof the purine nucleoside analog having the formula: ##STR9## wherein Ris selected from the group of substituent groups consisting of amino,hydroxyl, mercapto, methylmercapto and substituted amino NR'R" whereinR' and R" may be the same or different and are hydrogen, methyl, ethyl,propyl or phenyl, which method comprises:(A) preparing the intermediate##STR10## wherein X is halogen, by (1) hydrolysing2-azabicyclo[2.2.1]hept-5-en-3-one under acid conditions.(2) esterifyingthe carboxyl function of the resulting cis-4-aminocyclo-pent-2-enecarboxylic acid salt, (3) acetylating the amino group of the resultingester, (4) reducing and acetylating said ester, (5) epoxidizing theresulting acetate, (6) hydrolysing and acetylating the resultingepoxide, (7) forming an amine from the resulting (±)-4α-acetamido-2β,3α-diacetoxy-1α-cyclopentanemethyl acetate, and (8) condensing saidamine with 5-amino-4,6-dihalopyrimidine to produce said intermediate;(B) closing the ring of said intermediate by reaction with a ringclosing agent selected from the class consisting of diethoxymethylacetate and triethyl orthoformate, and (C) replacing the halogen with asubstituent from the group consisting of amino, hydroxyl, mercapto,methylmercapto and substituted amino.
 2. A method according to claim 1further characterized in that R is NH₂ and said halogen is replaced byreaction with ammonia.
 3. A method according to claim 1 furthercharacterized in that R is OH and said halogen is replaced by refluxingin aqueous acid.
 4. A method according to claim 1 further characterizedin that R is SH and said halogen is replaced by reaction with thiourea.5. A method according to claim 1 further characterized in that R isSCH₃, said halogen is replaced by a mercapto group by reaction withthiourea and said mercapto is methylated by reaction with methyl iodide.